There is considerable evidence that a subset of intestinal intraepithelial lymphocytes (IEL) develop extrathymically in mice, leading to the hypothesis that the intestine functions as a primary lymphoid organ in the development of these T cells. We, and others, have characterized cells found in lymphoid aggregates called, cryptopatches (CP) and T cell receptor negative (TCR-) cells, dispersed within the epithelium of mice, which have several phenotypic and functional characteristics of T cell progenitors. Nevertheless, the lineage relationships between these cells and the mechanisms by which putative progenitors develop into TCR+ IEL in vivo is presently unknown. Recent data in mice transgenic for the male, H-Y specific alphabeta T cell receptor (H-Y TCR), indicate that H-Y TCR+ IEL are present only in male mice, while female mice lack H-Y TCR+ transgenic IEL. This suggests that the development of IEL in male mice is antigen driven, while female mice fail to develop H-Y TCR+ IEL due to the lack of the cognate antigen. The central hypothesis of this proposal is H-Y TCR+ IEL arise via a distinct pathway of T cell development centered in the small intestine and that establishment of these T cells is self-antigen driven. We propose that bone marrow stem cells form cryptopatch aggregates early in neonatal life and that precursor IEL (pre-IEL) derived from these aggregates continue their differentiation within the intestinal epithelium establishing TCR+ IEL in response to antigen encountered in the gut. In the First Specific Aim we will investigate the origin of cells resident within cryptopatch aggregates and determine the dependence of these aggregates on the thymus and other key factors. In the Second Specific Aim we will determine the developmental potential of pre-IEL and the lineage relationships between these cells and TCR+ IEL. And finally, in the Third Specific Aim we will determine the requirements for selection and expansion of H-Y TCR+ IEL and determine the functional specificity of these cells. The studies in this application, are focused on the H-Y TCR transgenic mouse line. This system will allow us to define the developmental pathway and functional specificity of intestinal T cells driven by self-antigen reactivity in the gut. Defining the developmental pathways of T cells in the intestine will shed light on their role in immune homeostasis in the intestine and in the pathogenesis of inflammatory bowel disease.